A. SPECIFIC AIMS This research program combines expertise from three different laboratories at The Ohio State University and an industry partner to work on the first proof of concept for liposomal glycolipid drug delivery to dendritic cells for better harnessing the wide range of immunological functions of natural killer T cells. Conventional CD4+ and CD8+ T cells of the immune system recognize specific peptide antigens bound to major histocompatibility complex (MHC) class II or MHC class I molecules, respectively. In contrast, a specialized subpopulation of T cells called NKT cells recognize glycolipids antigens presented by the MHC class l-like molecule; CD1d. NKT cells represent a distinct population of T cells that express a conserved alpha-beta T cell receptor (TCR) and natural killer (NK) receptors. Mouse (m) Valpha14 and human (h) Valpha24 NKT cells regulate a number of critical biological conditions in vivo, including malignancy and infection, as well as autoimmune diseases, through the rapid secretion of T helper 1 (Th1) and Th2 cytokines and chemokines. A synthetic glycosphingolipid called alpha-galactosylceramide (alpha-GalCer), originally derived from a marine sponge, has been used in research as an exogenous ligand for CD1d to stimulate NKT cells. Moreover, alpha-GalCer is currently being tested in cancer patients. However, no complete objective response has been found in several clinical trials conducted by different teams in the United States, Japan and Europe. One of possible reasons might be that alpha-GalCer is too strong ligand that stuns the NKT cells to bring anergy. So far, a much more successful NKT cell immunotherapy approach is to re-induce human mature dendritic cells pulsed with alpha-GalCer ex vivo into patient to expand NKT cells. However, DCs are a small population of cells, and their isolation and pulsing with antigen can be impractical. Thus, we propose to incorporate alpha-GalCer (or other glycosphingosines) into liposomes to deliver the drug to DCs for optimal immunotherapy. Among many receptors on DCs for targeting, we choose recently discovered DC-specific intercellular adhesion molecule 3 (ICAM-3) grabbing non- integrin (DC-SIGN). Human DC-SIGN is abundantly expressed on DCs residing in lymphoid tissues and at mucosal surfaces, dermal DCs, and by specialized macrophages in placenta and lung. Several laboratories have demonstrated that targeting DC-SIGN by anti-DC-SIGN antibody afford the best strategy to deliver antigens or drug into DCs. High mannose glycans are the ligand for DC-SIGN, but recently it was discovered that DC-SIGN bind the second class of glycans: fucosyl containing oligosaccharides. Thus, this proposed research program will be the first attempt to explore the anti-tumor effect of NKT ligands by targeting the glycosphingosine drug specifically to dendritic cells with liposomes which display fucose containing oligosaccharides such as Le-a and Le-x. Four specific aims are proposed in this program: 1. Synthesis of Le-a and Le-x oligosaccharides, their tetramers and their conjugates with lipids, measurement of their binding with DC-SIGN. 2. Production of two liposomal systems. System A consists of a-GalCer with anti-DC-SIGN antibody displayed; system B consists of alpha-GalCer with fucosylated oligosaccharide displayed (e.g. Le-a and Le-x). 3. Investigation in vitro of the two liposomal systems for their targeting and cytotoxicity. 4. Investigation in vivo of the two liposomal systems for their antitumor effects in mouse models. This exploratory research will open many future possibilities on NKT activation, dendritic cell targeting, and liposomal technology. The proposed DC-targeted liposomal systems incorporated with glycolipids as NKT cell activators can be potentially used in vaccine adjuvant development and in preventing infections, autoimmune diseases and inflammatory diseases. This work will set up a novel platform to better use a wide range of possible medical benefits brought about by our recent understanding on the immunoregulatory and immunomodulatory functions of natural killer T cells. [unreadable] [unreadable] [unreadable]